Presentation on theme: "SUPPLEMENTARY FIGURES Figure S-I. Fluorescence intensity and subcellular localization of transfected EGFP fusion proteins HeLa cells were transferred into."— Presentation transcript:
SUPPLEMENTARY FIGURES Figure S-I. Fluorescence intensity and subcellular localization of transfected EGFP fusion proteins HeLa cells were transferred into 96-well plates (Costar) at 5 x 10 3 /well each, incubated for 24 h and then transfected with EGFP, EGFP-importin 2 full-length and EGFP-importin 2 C-mutant constructs using Effectene Transfection Regent (Qiagen). To quantitate cytoplasmic and nuclear fluorescence, images were captured on an ArrayScan VTI instrument (Thermo Fisher) and analysed using Cellomics software and the Compartmental Analysis Bioapplication. The analysis algorithm uses DAPI fluorescence to define a mask used to measure nuclear fluorescence. The region for whole cell measurements is generated by dilating the mask to cover as much of the cytoplasm as possible without going outside the cell boundary. The cytoplasm region is generated by removal of the nuclear from whole cell regions to create a ring. Average pixel intensities of EGFP within these regions were calculated. Non-transfected cells (with autofluorescent pixel intensity similar to non-transfected controls) were gated out of the analysis. Three independent experiments were done, and 300 cells were measured for each transfection. One of the independent results was presented here. Figure S-II. Validation of down-regulated genes by quantitative PCR HeLa cells were transfected with EGFP as a control, EGFP-importin 2 full-length or C-mutant constructs, and mRNA expression levels of indicated genes were analyzed by qPCR. The results were from three independent experiments and were presented in comparison to values in the EGFP-expressing cells as the mean ± SEM (n=3 each). **p<0.01; Student s t-test. Primers are shown in Supplemental Table S-III. Figure S-III. Validation of ChIP analysis (A) HeLa cells were exposed to 200 μM H 2 O 2 for 30 min and subjected to immunoprecipitation using an anti-importin 3 antibody in accordance with ChIP assay. The bound proteins were analyzed by Western blotting using anti-importin 3 antibody. (B) HeLa cells were exposed to 200 μM H 2 O 2 for 30 min and subjected to immunoprecipitation using an anti- p53 antibody. After DNA purification from the precipitated the chromatin sample, PCR was performed to amplify sequences in the indicated promoter regions. Figure S-IV. Establishment of STK35 expressing stable cell lines and knock-down experiments by RNAi oligonucleotides (A) 293F cells were transfected with wtag, wtag-STK35S or wtag-STK35L1 and established stable cell lines were selected by single cell clones. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS-PAGE and detected by Western blotting using anti-GST antibody. The band provided with asterisk is suspected to be a degradation product of STK35L1. (B) 293F stably expressing wtag as a control (Cont.), wtag-STK35S or wtag-STK35L1 were pretreated for 30 min in the absence or presence of 50 μM zVAD-fmk and exposed to 2 mM H 2 O 2 for 2 h or 0.5 μM STS for 24 h, respectively. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS- PAGE and analyzed by Western blotting for PARP, and GAPDH. (C) 293F cells stably expressing wtag as a control (Cont.), wtag-STK35S or wtag-STK35L1 were exposed to 0.5 μM STS for 4 h. Cells were stained with PI and then sorted by FACS. Values are means ± SEM (n=3 each) of PI-positive cells. (D) HeLa cells were transfected with Luciferase siRNA (siLUC.) as a control or STK35 siRNA oligonucleotides (siSTK35-1 or siSTK35-2) and incubated for 48 h. The mRNA expression levels of STK35 were analyzed by qPCR. The results are from three independent experiments and are presented in comparison to the values in control siRNA-expressing cells as the mean ±SEM (n=3 each). **p<0.01; Student s t-test.
EGFP-importin 2 Full EGFP-importin 2 C-mut 4 Total intensity (log 10 ) N/C ratio Total intensity (log 10 ) Total intensity (log 10 ) EGFP Figure S-I N/C ratio
Input Normal IgG (Rabbit) Anti-p53 p21 STK35 B Importin 3 IgG heavy chain kDa Input Normal IgG (Goat) Anti-importin 3 A Figure S-III
Cleaved PARP GAPDH Cont.STK35SSTK35L1 Untreated H2O2H2O2 H 2 O 2 + z-VAD-fmk STS + z-VAD-fmk Cont.STK35SSTK35L1Cont.STK35SSTK35L1Cont.STK35SSTK35L1Cont.STK35SSTK35L1 B Figure S-IV % PI positive cells Cont. STK35S STK35L1 STSUntreated C wtag wtag-STK35S wtag-STK35L1 kDa * A Relative STK35 mRNA level ** siSTK35-2 ** siSTK35-1 siLUC. D
a) Fold change ( or 2-fold) indicates the ratio of microarray signals in the EGFP-importin 2 full-length gene expressing cells compared to those in the control EGFP transfected cells. b) Fold change ( or 2-fold) indicates the ratio of microarray signals of the EGFP-importin 2 C-mutant gene expressing cells to those of the control EGFP transfected cells. c) Partial molecular function/Characterization is provided according to the PANTHER classification system (http://www.pantherdb.org/) d) Abbreviations; N: Nucleus, C: Cytoplasm, M: Membrane, NM: Nuclear Membrane, PM: Plasma Membrane, Ce: Centrosom, Mit: Mitochondria, S: Secreted, G: Golgi, ER: Endoplasmic Reticulum, AJ: Adherents Junctions, CG: Cytoplasmic granular Table S-I. Summary of up- or down-regulated genes in microarray analysis
Table S-II. Summary of replication-dependent histone genes
Table S-III. Primers for qPCR
Broad Institute, Cambridge, MA, USA (http://www.broadinstitute.org/gsea/index.jsp) a) number of genes in a particular gene set b) enrichment score c) normalized enrichment score d) nominal pvalue e) false discovery rate * these gene sets were below a NOM p-val of 0.05 and a FDR q-val of 0.25 FullUp-regulated * Down-regulated C-mutUp-regulated Down-regulated CHR21Q22 CHR1P36 CHR14Q24 CHR8Q24 CHR6P21 CHR19P13 CHR1Q42 CHR16P13 CHR20Q11 CHR20Q13 CHR10Q26 CHR14Q32 CHR9Q34 CHR14Q24 CHR21Q22 CHR10Q26 CHR8Q24 CHR20Q11 CHR19P13 CHR6P21 CHR9Q34 CHR16P13 CHR20Q13 CHR1Q42 CHR1P36 CHR14Q32 Gene Set Name a) Size b) ES c) NES d) NOM p-val e) FDR q-val Table S-IV. GSEA tests for enrichment of MSigDB C1 gene sets Analysis of the expression data was performed using the GSEA software (Broad Institute, Cambridge, MA, USA). GSEA examines ranked lists of genes for enrichment of biological pathways contained within datasets for different databases of MSigDB: C1 (positional gene sets), C2 (curated gene sets), C3 (motif gene sets), C4 (computational gene sets), and C5 (GO gene sets).
* * * FullUp-regulatedHSC_HSCANDPROGENITORS_ADULT HSC_HSCANDPROGENITORS_FETAL HSC_HSCANDPROGENITORS_SHARED BRCA_ER_NEG VHL_NORMAL_UP STEMCELL_HEMATOPOIETIC_UP ALZHEIMERS_DISEASE_UP STEMCELL_NEURAL_UP Down-regulatedRUTELLA_HEMATOGFSNDCS_DIFF HSC_EARLYPROGENITORS_ADULT CMV_HCMV_TIMECOURSE_ALL_DN HSC_EARLYPROGENITORS_FETAL HSC_EARLYPROGENITORS_SHARED POD1_KO_DN LEE_TCELLS2_UP HSC_LATEPROGENITORS_FETAL HSC_LATEPROGENITORS_ADULT HSC_LATEPROGENITORS_SHARED BRCA_ER_POS RCC_NL_UP STEMCELL_EMBRYONIC_UP AGEING_KIDNEY_UP ALZHEIMERS_DISEASE_DN C-mutUp-regulatedHSC_HSCANDPROGENITORS_ADULT HSC_HSCANDPROGENITORS_FETAL HSC_HSCANDPROGENITORS_SHARED BRCA_ER_NEG STEMCELL_NEURAL_UP STEMCELL_EMBRYONIC_UP VHL_NORMAL_UP STEMCELL_HEMATOPOIETIC_UP RCC_NL_UP ALZHEIMERS_DISEASE_DN Down-regulatedRUTELLA_HEMATOGFSNDCS_DIFF CMV_HCMV_TIMECOURSE_ALL_DN BRCA_ER_POS HSC_LATEPROGENITORS_FETAL HSC_EARLYPROGENITORS_ADULT HSC_LATEPROGENITORS_ADULT HSC_LATEPROGENITORS_SHARED ALZHEIMERS_DISEASE_UP POD1_KO_DN LEE_TCELLS2_UP HSC_EARLYPROGENITORS_SHARED HSC_EARLYPROGENITORS_FETAL AGEING_KIDNEY_UP Table S-IV. GSEA tests for enrichment of MSigDB C2 gene sets Gene Set Name a) Size b) ES c) NES d) NOM p-val e) FDR q-val
Gene Set Name a) Size b) ES c) NES d) NOM p-val e) FDR q-val FullUp-regulated None Down-regulated GCM_NF2 C-mut Up-regulated GCM_NF2 Down-regulated None Table S-IV. GSEA tests for enrichment of MSigDB C4 gene sets Gene Set Name a) Size b) ES c) NES d) NOM p-val e) FDR q-val FullUp-regulated None Down-regulated None C-mutUp-regulated None Down-regulated None Table S-IV. GSEA tests for enrichment of MSigDB C5 gene sets